Description of Related Art
Transcription of DNA into mRNA is regulated by the promoter region of the DNA. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA. RNA polymerases from different species typically recognize promoter regions comprised of different sequences. In order to express a protein-encoding gene in a host cell, either the promoter driving transcription of the protein-encoding gene must be recognized by a host RNA polymerase, or an RNA polymerase which recognizes the promoter driving transcription of the protein-encoding gene must be provided to the host cell (U.S. Pat. No. 6,218,145).
There are many reasons for which it is beneficial to obtain cDNA and transcribe one or more target nucleic acid sequences. For example, in vitro transcription is frequently used in methods to make probes corresponding to mRNA sequences in samples in order to profile the expression of genes in cells of a particular type versus another type or versus a similar type in response to different conditions or stimuli. Contemporary gene expression profiling is typically performed by simultaneously hybridizing labeled probes prepared from one or more samples to arrays or microarrays having sequences for up to hundreds or thousands of different genes attached to a surface. In some cases, the expression or lack of expression of particular genes may correlate with or be indicative of the presence or status of a disease state, such as, but not limited to, a cancer.
Examples of methods that involve in vitro transcription for making probes for gene expression profiling are described in: Murakawa et al., DNA 7:287-295, 1988; Phillips and Eberwine, Methods in Enzymol. Suppl. 10:283-288, 1996; Ginsberg et al., Ann. Neurol. 45:174-181, 1999; Ginsberg et al., Ann. Neurol. 48:77-87, 2000; VanGelder et al. Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990; Eberwine et al., Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992; U.S. Pat. Nos. 5,021,335; 5,168,038; 5,545,522; 5,514,545; 5,716,785; 5,891,636; 5,958,688; 6,291,170; and PCT Patent Applications WO 00/75356 and WO 02/065093.
Still other methods use in vitro transcription as part of a process for amplifying and detecting one or more target nucleic acid sequences in order to detect the presence of a pathogen, such as a viral or microbial pathogen, that is a causative agent for a disease or to detect a gene sequence that is related to a disease or the status of a disease for medical purposes. Examples of methods that use in vitro transcription for this purpose include U.S. Pat. Nos. 5,130,238; 5,194,370; 5,399,491; 5,409,818; 5,437,990; 5,466,586; 5,554,517; 5,665,545; 6,063,603; 6,090,591; 6,100,024; 6,410,276;.Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173, 1989; Fahy et al, In: PCR Methods and Applications, pp. 25-33, 1991; PCT Patent Application Nos. WO 89/06700 and WO 91/18155; and European Patent Application Nos. 0427073 A2 and 0427074 A2.
Most DNA-dependent RNA polymerases read double-stranded DNA, limiting RNA synthesis to systems in which a double-stranded DNA template is available. The synthesis of RNA using single-stranded DNA is not as common. All of the methods referenced above for making probes for gene expression profiling or for amplifying and detecting one or more target nucleic acid sequences require, at a minimum, the use of a double-stranded transcription promoter, and in most cases, also require the use of a double-stranded DNA as a template. Synthesizing RNA using a single-stranded DNA template immobilized on a solid support is described in U.S. Pat. No. 5,700,667, but transcription of the single-stranded template still required formation of a double-stranded promoter region for binding of the RNA polymerase.
In contrast to the methods in the art, the present invention provides methods, compositions and kits for transcription of target nucleic acid sequences using RNA polymerases that bind single-stranded DNA promoters and read single-stranded DNA templates.